AFLP Markers Differentiate Isolates of Colletotrichum gossypii from C. gossypii var. cephalosporioides* Renata Silva-Mann1**, Maria G. G. Carvalho Vieira2, José C. Machado3, José Roberto Bernardino Filho2, Kalinka C. C. Salgado2 & Mikel R. Stevens4 1Departamento de Eng. Agronômica, Universidade Federal de Sergipe, Cidade Universitária Prof. José Aloísio de Campos, Av. Marechal Rondon s/n, Jardim Rosa Elze, CEP 49100-000, São Cristóvão, SE, e-mail: [email protected]; 2 Laboratório de Análise de Sementes-DAG/UFLA, e-mail: [email protected]; 3Laboratório de Patologia de Sementes-DFS/ UFLA, e-mail: [email protected], Cx. Postal 37, CEP 37200-000, Lavras, MG; 4Department of Plant and Animal Sciences, Brigham Young University, Provo, UT, USA e-mail: [email protected] (Accepted for publication on 08/07/2004) Corresponding author: Renata Silva-Mann SILVA-MANN, R., VIEIRA, M.G.G.C., MACHADO, J.C., BERNARDINO FILHO, J.R., SALGADO, K.C.C. & STEVENS, M.R. AFLP markers differentiate Colletotrichum gossypii from C. gossypii var. cephalosporioides. Fitopatologia Brasileira 30:169-172. 2005. ABSTRACT Fungal diseases in cotton (Gossypium hirsutum), such as anthracnose caused by Colletotrichum gossypii and ramulose caused by C. gossypii var. cephalosporioides, are responsible for large yield losses. These pathogens are seed borne and morphologically similar although they induce different symptoms, which can lead to misdiagnosis using the blotter testing method. The present study was carried out to assess the viability of using Amplified Fragment Length Polymorphism (AFLP) markers to differentiate these pathogens. Five isolates, for each pathogen, were classified according to pathogenicity on cotton plants, and mycelial growth morphology. Conidial suspensions were sprayed on 30-day-old cotton plants and the symptoms assessed ten and 40 days after inoculation. For growth morphology 200 cottonseeds were inoculated with seven-day-old pure cultures, and the mycelial traits observed under a stereoscopic microscope seven days after inoculation. The DNA for AFLP analysis was obtained from seven-day-old fungal mycelia grown in liquid medium, using the Dneasy Qiagen protocol. Using the AFLP technique 318 polymorphic bands were selected to estimate similarities using Dice’s Coefficient. The results clearly distinguished between ramulose and anthracnose isolates, which agreed with morphological and pathogenicity testing. Additional keywords: molecular markers, anthracnose, ramulose, cotton diseases, seed borne pathogens. RESUMO Marcadores AFLP diferenciam Colletotrichum gossypii de C. gossypii var. cephalosporioides Doenças fúngicas em algodoeiro (Gossypium hirsutum), tais como antracnose causada por Colletotrichum gossypii e ramulose causada por C. gossypii var. cephalosporioides, levam a grandes perdas em produtividade. Estes microrganismos são patógenos veiculados por sementes e morfologicamente similares apesar deles induzirem sintomas diferentes, que podem levar a um erro no diagnóstico usando o teste de sanidade com sementes em rolo de papel. O presente estudo foi realizado para acessar a viabilidade do uso de marcadores Amplified Fragment Length Polymorphism (AFLP) para diferenciar estes patógenos. Cinco isolados, para cada patógeno, foram classificados de acordo com a patogenicidade e morfologia de crescimento micelial. As suspensões de conídios foram pulverizadas em plantas de algodoeiro com 30 dias e os sintomas avaliados aos dez e 40 dias após a inoculação. Para a morfologia de crescimento micelial, 200 sementes foram inoculadas com culturas puras com sete dias, e as características miceliais observadas sob microscópio esterioscópico sete dias após a inoculação. O DNA para a análise de AFLP foi obtido de micélio fúngico com sete dias, crescido em meio líquido, usando o protocolo Dneasy Qiagen. A técnica AFLP produziu 318 bandas polimórficas que foram selecionadas para calcular as similaridades usando o Coeficiente de Dice. Os resultados claramente distinguiram os isolados causadores de ramulose dos de antracnose, os quais concordam, com dados morfológicos e de patogenicidade. Additional keywords: marcadores moleculares, antracnose, ramulose, doenças do algodoeiro, patógenos de sementes. Fungal infections account for large crop losses, hirsutum L.) seeds in Brazil, along with the description of the especially in the Brazilian rainy season. Studies have identified effects of pathogenic fungi on seed germination and disease a number of fungal species associated with cotton (Gossypium transmission to virgin cotton lands (Teixeira, 2001). Anthracnose caused by Colletotrichum gossypii South, causes *Part of the Doctor Thesis of the first author. Universidade Federal de Lavras. (2002). lesions on all parts of the cotton plant, while ramulose is caused **CAPES fellowship by C. gossypii (South) var. cephalosporioides A. S. Costa is Fitopatol. bras. 30(2), mar - abr 2005 169 R. Silva-Mann et al. the most severe cotton disease in Brazil (Davis et al., 1981). (Model MA 140) at 170 rpm. The mycelia were filtered and Anthracnose is mainly a seedling disease that causes “damping lyophilized for 48 h at -40 oC and 10-1 mbar pressure. Total off”. The two pathogens cannot be distinguished using the DNA extraction was performed using the DNeasy Qiagen Kit. paper-blotting test. Therefore, this research was conducted to Digestion of genomic DNA was performed in 1.5 ml assess the viability of utilizing the Amplified Fragment Length tube with 2.5 ìl of DNA (10ng/ìl), 5.0 ìl of 5X buffer reaction Polymorphism (AFLP) marker technique (Zabeau, 1993) to (Life Technologies – Gibco BRL), 4 U/µl of MseI, 10 U/µl distinguish C. gossypii from C. gossypii var. cephalosporioides. EcoRI, and 25 ìl of de-ionized water. The mixture was Inoculum was prepared by growing Colletotrichum incubated for two h at 37 °C and placed at 70 °C to inactivate spp. isolates (Table 1) on potato-dextrose-agar (PDA) medium the enzyme reaction. Once inactivation was complete 24 ì l for seven days. Inoculations were carried out by spraying 30- of adaptors solution (Life Technologies – Gibco BRL) was day-old plants of IAC-22, ITA-90 (CNPA Itamarati 90), ITA- added to the digestion along with T4 DNA ligase (3 U/µl). 96 (CNPA Itamarati 96), Redenção (Epamig-4) and Precoce The cocktail was incubated at 20 °C for 12 h then diluted to (Epamig-5) cultivars with a 106 conidia/ml suspension. 0.1 X with TE, and stored at -20 °C. Sterilized de-ionized water was utilized as a control. Pre-amplification reactions were accomplished by Inoculated plants were kept in a moist chamber for 72 h. adding 5 µl of 10X PCR buffer, 1 U of Taq DNA polymerase Disease assessment was carried out ten and 40 days after for a final volume of 51 ì l . The PCR reaction was performed inoculation, following a modified version of the disease in Perkin-Elmer thermocycler (model 9600), using 94 °C for severity scale of Cia (1977) for anthracnose and ramulose, as 30 min, 56 °C for 60 s, and 72 °C for 60 s, for 20 cycles. follows: (1) Plant without symptoms (no lesions); (2) Plant The primers used were MseI (GATGAGTCCTGAGT with star spots on the top leaves; (3) Plant with shortened AA) and EcoRI (GACTGCGTACCAATTCCAA). The primer internodes (to as low as 60% of the control) and with circle combinations were: (1) EcoRI AT- MseI CG, (2) EcoRI AT – and star leaf spots on the top leaves; (4) Plant with over MseI CA, (3) EcoRI AT – MseI CC, (4) EcoRI AA – MseI CA, sprouting and reduced internodes of 40 to 60% of the controls; (5) EcoRI AA – MseI CC, (6) EcoRI AA – MseI CT, (7) EcoRI (5) Plant with over sprouting and reduced growth, reduced AA – MseI CG. The primers were labeled with 33P in 50 ì l internodes less than 60% of the control. reactions containing 0.1 ml of a 50 ng/ml of EcoRI primer + Cottonseeds (cultivars IAC-22 e ITA-90) were two base pairs, 10ìl of de-ionized water, 10 ìl of 5X kinase 33 inoculated on seven-day-old pure cultures of C. gossypii and buffer, 10 ìl of ãP (3.000 Ci/mmol), 0.2 U of T4 kinase. C. gossypii var. cephalosporioides. Seven days after The reagents were mixed and incubated for one hour at 37 inoculation the blotter test (Brasil, 1992) was performed using °C. At the end of the reaction time the mixture was inactivated a random experimental design with four replications of 50 at 70 °C. seeds each, and the mycelial traits observed under a Amplification reactions were performed using 50 ng/ stereoscopic microscopy (Tanaka et al., 1996). ml of EcoRI and of MseI primer, 5 mM of dNTPs, for a final For AFLP analysis, mycelia from monosporic cultures volume of 50 ì l . Added to another tube were 79 ìl of de- were obtained by cultivating isolates in liquid medium (10 g ionized water, 20 ìl of PCR buffer 10X, and DNA Taq glucose; 1.0 g de NH4H2PO4; 0.2 g KCl; 0.2 g MgSO4. 7 H2O; polymerase (5 units/ìl ) for a total volume of 100 ìl. For 5 g yeast extract; 1 ml CuSO4. 5 H2O; 5% polyvinyl-pyrrolidone; amplifications, 5 µl of the pre-amplification reaction (diluted 1 ml ZnSO4: 7 H2O. 1% p/v for 1000 ml sterile distilled water) 1:50 with TE buffer) were used. The reactions were performed (Mills et al., 1994) for seven days at 22ºC in orbital shaker in a Perkin-Elmer thermocycler using 94 °C for 30 s, 65 °C for 30 s, and 72 °C for 60 s. The thermocycler was programmed for 12 cycles with a 0.7 °C decrease in the TABLE 1 - Fungus isolates1 used in this study, disease symptoms annealing temperature after each cycle, followed by 23 cycles and origin of 94 °C/30 s, 56 °C/30 s and 72 °C/60 s. Isolate Disease name Host Local After amplification, 20 ìl of formamide mix (98% CGC1 Ramulose2 Gossypium hirsutum Piracicaba-SP formamide, 10 mM EDTA, 1% bromophenol blue and 1% 10166 Ramulose G.